Ezh2 emerges as an epigenetic checkpoint regulator during monocyte differentiation limiting cardiac dysfunction post-MI

Epigenetic regulation of histone H3K27 methylation has recently emerged as a key step during alternative immunoregulatory M2-like macrophage polarization; known to impact cardiac repair after Myocardial Infarction (MI). We hypothesized that EZH2, responsible for H3K27 methylation, could act as an epigenetic checkpoint regulator during this process. We demonstrate for the first time an ectopic EZH2, and putative, cytoplasmic inactive localization of the epigenetic enzyme, during monocyte differentiation into M2 macrophages in vitro as well as in immunomodulatory cardiac macrophages in vivo in the post-MI acute inflammatory phase. Moreover, we show that pharmacological EZH2 inhibition, with GSK-343, resolves H3K27 methylation of bivalent gene promoters, thus enhancing their expression to promote human monocyte repair functions. In line with this protective effect, GSK-343 treatment accelerated cardiac inflammatory resolution preventing infarct expansion and subsequent cardiac dysfunction in female mice post-MI in vivo. In conclusion, our study reveals that pharmacological epigenetic modulation of cardiac-infiltrating immune cells may hold promise to limit adverse cardiac remodeling after MI.


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None of the findings in this article are relevant to patient sex and/or gender All information relevant to the patients characteristics are provided as supplementary information in supplementary tables 2 and 5 Between Nov 2018 and June 2020, the EPICAM prospective study enrolled 48 patients at Rouen University Hospital after diagnosis based on coronary angiography procedure. According to French legislation, all patients read the information note detailing the study protocol. All patients orally consented to the collection of an additional volume of blood during their usual care as well as the processing of their personal data (such as age, sex and coronary diagnosis) prior to the blood sampling. Inclusion criteria were: 1) age greater than or equal to 18 years and 2) admission to hospital for a coronary angiography. The exclusion criteria were 1) Infectious diseases, 2) Current pregnancy or breastfeeding, 3) Obesity (BMI > 30 kg/m²), 4) Hematological pathologies, 5) Anemia and 6) Inflammatory and autoimmune pathologies. As this is a monocentic study with a different number of patients included in each group, selection bias may be present and migth have impacted the results.
Sample size were chosen according to the experimental experience of the procedures in the laboratory (PMID: 32404007, 27059805, 24760754). Regarding Human research participants, the sample size was determined by the duration of the recruitment period in agreement with the duration of the study approved by the CPP Ile de France V.
Data were excluded based on the identification of outliers provided by GraphPad Prism 8 software Reproducibility of experiment was assessed by reproducing at least 3 three times every single experiment For Human research study, participant were not randomized but separately included in the study in double blind manner. Randomization was not relevant to this study as patients were included in one of the three groups based on a diagnosis obtained after coronary angiography procedure. For mouse in vivo study, animals were randomized after surgical procedure before receiving the pharmacological treatment.
Chromatin Immunoprecipitation: 2 µg of the following antibodies were used: rabbit anti-H3K4me3 (Millipore, Cat#07-473), rabbit anti-H3K27me3 (Millipore, Cat#07-449), or normal rabbit IgG (Millipore, Cat#12-370). Specificity and Specificity and sensibility for each of the primary antibodies used for each application has been validated in the laboratory based on advised concentration or dilution provided by the manufacturer at first and refined using appropriate positive and negative controls relevant to each application. For instance anti-CD206 specificity and sensibility for Immunohisto/cytochemistry was assessed with cell autofluorescence control as well as cell negative control (cells from the same cell lineage not expressing CD206, i.e. M0 and M1 macrophages) and cell positive control (cells from the same cell lineage expressing CD206, i.e. M2 macrophages). Similar types of procedure were applied for flow cytometry (use of (use of IgG Isotype controls, compensation matrix, cell positive and negative controls), for western blotting (with additional sensitivity assay following sequential increased protein loading), and Chromatin Immunoprecipitation (use of IgG Isotype controls, background or signal level measurement on well-known negative and positive genomic regions respectively as described in Figures 3a, 3c and 5).for each of the primary antibodies used for each application has been validated in the laboratory based on advised concentration or dilution provided by the manufacturer at first and refined using appropriate positive and negative controls relevant to each application. For instance anti-CD206 specificity and sensibility for Immunohisto/cytochemistry was assessed with cell autofluorescence control as well as cell negative control (cells from the same cell lineage not expressing CD206, i.e. M0 and M1 macrophages) and cell positive control (cells from the same cell lineage expressing CD206, i.e. M2 macrophages).
Monocytes were directly or indirectly (after red blood cell lysis (eBioscience Cat#00-4333-57) isolated by negative selection using EasySep™ monocyte isolation kits for human (male and female, using StemCell Technologies Cat#19669) and mouse (female, using StemCell Technologies Cat#19861) cells from fresh peripheral blood samples according to the manufacturer's instructions. TIB-204™ (WEHI-265.1) mouse monocyte cell line (ATCC Lot#4249478) was used in this study.
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No use of wild animals
We only used female mice for MI studies as they display lower mortality than male in the MI model, which help us reduce the numbers of animals included in our studies. In addition, females express both alleles of the H3K27me3 demethylase Kdm6a gene encoding the Utx protein.
No field collected samples were used in the study All animal experiments performed in this study were approved by the regional ethics review board (CENOMEXA) and the french Ministère de l'Enseignement Supérieur et de la Recherche, in line with E.U and French legislation, referred as APAFIS #8157-2016121311094625-v5 Normandy, APAFIS #31897-2021111911125883 v4. This is a non interventional protocol registered as RCB: 2018-A02108-47 This is an internal non interventional protocol to the University Hospital of Rouen Between Nov 2018 and June 2020, the EPICAM prospective study enrolled 48 patients at Rouen University Hospital. The CPP Ile de France V has approved the study august 10th 2018 (RCB: 2018-A02108-47). All patients read the information sheet and accepted to participate to the study before blood collection. Inclusion criteria were: 1) age greater than or equal to 18 years and 2) admission to hospital for a coronary angiography. The exclusion criteria were 1) Infectious diseases, 2) Current pregnancy or breastfeeding, 3) Obesity (BMI > 30 kg/m²), 4) Hematological pathologies, 5) Anemia and 6) Inflammatory and autoimmune pathologies. During the coronarography procedure, peripheral blood was collected into 4 BD Vacutainer® EDTA K2 tubes (Becton Dickinson Cat#367862). In-hospital data were entered into a dedicated database.
The objective of this non interventional protocol was to study the whole transcriptomic profile human monocytes in patients assesed by mRNA-seq and RTqPCR methods.